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| Author(s): |
Loiane Sampaio Tavares
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Pirassununga. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT) |
| Defense date: | 2018-12-14 |
| Examining board members: |
Ricardo Luiz Moro de Sousa;
Mateus Maldonado Carriero;
Danielle de Carla Dias;
Marcio Hipolito
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| Advisor: | Ricardo Luiz Moro de Sousa |
| Abstract | |
The genus Ranavirus, especially the Frog virus 3 (FV3) species, is considered a growing threat to amphibian populations in various parts of the world, triggering outbreaks that often result in mass mortality and substantial economic losses. In this sense, it was proposed the isolation of a pathogenic FV3-like strain associated with an outbreak with high mortality of adult amphibians (Lithobates catesbeianus) in a frog farm in the State of São Paulo, Brazil, as well as the molecular and phenotypic characterization of the isolated virus. In addition, we aimed to verify the possible induction of the mechanism of cell death by apoptosis by this strain. Virus isolation was performed from organ fragments of animals that died, which were inoculated into BF-2 cells (Lepomis macrochirus). The polymerase chain reaction (PCR) technique was conducted with specific primers and directed to two highly conserved genome regions of the ranavirus: MCP and 53R. Partial sequencing of the gene encoding the major capsid protein (MCP) was performed, followed by alignment and phylogenetic analysis with other ranaviruses. Other diagnostic techniques, including transmission electron microscopy, indirect immunofluorescence and Western blot were used in the characterization and confirmation of the isolation. Two apoptotic markers were used to investigate the possible activation of apoptosis in infected BF-2 cells and sampled from 4 to 16 hours, including the activation of effector caspases and the fragmentation of cellular DNA by the TUNEL assay. We obtained the isolation of an FV3-like strain with typical cytopathic effects for ranavirus. PCR confirmed the presence of viral DNA in cultures of infected BF-2 cells, with positive results for MCP and 53R oligonucleotides. Analysis of the nucleotide sequence obtained for MCP revealed high homology (99%) with Frog virus 3, type species member of the genus Ranavirus (family Iridoviridae) and, in phylogenetic reconstruction, the isolated strain showed to be closely related to other ranaviruses detected in Brazil. Electron micrographs showed icosahedral particles in infected BF-2 cells, with a nucleocapsid measuring about 150 ηm, similar to ranaviruses. In the indirect immunofluorescence assay, BF-2 cells infected with the FV3-like isolate showed positive immunofluorescence labeling for MCP, whereas MCP was demonstrated by a Western blot assay, where we observed a polypeptide with a molecular mass estimated at 50 kDa. Finally, we verified that the FV3-like isolate is able to induce apoptosis in BF-2 cells, since effector caspases were detected at all experimental times, suggesting to be a caspase-dependent mechanism. The fragmentation of cellular DNA, clearly observed at all experimental times, confirmed the induction of apoptosis by the Brazilian FV3-like strain. The results obtained here become the basis for several future studies, and may contribute as a subsidy to better understand the outbreaks caused by these viruses in the country. (AU) | |
| FAPESP's process: | 16/25506-2 - Analysis of gene expression in the innate immune response in cell lines of fish origin triggered by Brazilian strain frog virus 3-like |
| Grantee: | Loiane Sampaio Tavares |
| Support Opportunities: | Scholarships in Brazil - Master |